Factor VIII is a protein involved in clotting blood and plays a critical role in hemostasis. The lack of factor VIII leads to the bleeding disorder hemophilia A. Hemostasis is maintained by an intricate and complex regulation of both coagulation and anti-coagulation pathways in the blood. The clotting cascade begins when cell damage activates the enzyme factor XII and ends when soluble fibrinogen is converted into fibrin by thrombin. The pathway involves the interaction of many proteolytic enzymes, e.g., factors XII, XI, IX, X and prothrombin, as well as numerous co-factors such as factors VIII and V. Once coagulation is initiated, the response is amplified by a cascade of protease activation steps that occur on the surface of endothelial cells and platelets. At each step an inactive protein is converted into a protease which in turn activates the next protein in the pathway. The cascade includes both positive-feedback and negative-feedback loops. The clotting pathway eventually leads to the formation of insoluble fibrin which, together with platelets, obscures blood flow at the site of tissue damage.
Certain steps of the pathway are accelerated by co-factors, such as factors VIII and V. Approximately 85 percent of hemophiliacs lack factor VIII; the remainder lack factor IX. Thrombin not only activates factors VIII and V, but it also can deactivate them, i.e., by activating protein C. The exact mechanisms by which the levels and activities of active factor VIII are controlled are still not completely understood.
Traditionally, hemophiliacs were treated with transfusions of whole blood. More recently, treatment has been with preparations of factor VIII concentrates derived from human plasma. However, the use of plasma-derived product exposes hemophilia patients to the possible risk of virus-transmissible diseases such as hepatitis and AIDS. Costly purification schemes to reduce this risk increases treatment costs. With increases in costs and limited availability of plasma-derived factor VIII, patients are treated episodically on a demand basis rather than prophylactically. Moreover, factor VIII typically exhibits limited stability after activation with thrombin, necessitating administration of large amounts of protein during a bleeding episode. Recombinantly produced factor VIII has substantial advantages over plasma-derived factor VIII in terms of purity and safety, as well as increased availability. Procoagulant proteins with enhanced stability are desirable to minimize the amount of protein infused into a patient during a bleeding episode. Accordingly, much research effort has been directed towards the development of recombinantly produced factor VIII.
In light of the known immunogenicity of plasma-derived factor VIII, one of the goals in developing new recombinant forms of factor VIII for use as a therapeutic agent is the development of products that do not induce an immune response. Approximately 15 percent of all hemophiliacs develop an immune response to factor VIII replacement therapy at some time during their lives. The resultant antibody production causes inhibition of subsequently infused factor VIII products and creates a difficult therapeutic situation for the patient. Attempts have been made, therefore, to develop recombinant forms of factor VIII that are modified in ways that reduce or eliminate such an immune response. It is not a priori possible, however, to predict in advance whether newly developed recombinant forms also result in the generation of new epitopes that, although absent from natural factor VIII preparations will themselves generate undesirable antibody responses.
Other goals in developing new, more stable recombinant forms of factor VIII include the introduction of specific mutations into factor VIII to gain an understanding of specific requirements for factor VIII activity, as well as the specific requirements for thrombin activation and subsequent inactivation of factor VIII. Because of the complex subunit structure and labile nature of factor VIII, and of its activated derivatives, study of the structure-function relationship has been difficult.
The domain, structure, and processing of factor VIII is set forth in detail in Kaufman, Nature 342:207(1989). The parent single chain precursor is 2351 amino acids long and has a domainal sequence of: EQU NH.sub.2 -A1-A2-B-A3-C1-C2-COOH
The full-length nucleotide sequence and corresponding amino acid sequence is set forth in SEQ ID NO: 1 and 2.
Upon secretion, a portion of the B domain is removed to generate an amino terminal-derived heavy chain (200 kD) and a carboxyl terminal-derived light chain (80 kD). The heavy chain is: NH.sub.2 -A1-A2-B and the light chain is: A3-C1-C2-COOH. There is a metal ion-dependent association between the two chains. In plasma, factor VIII circulates as an inactive co-factor which requires cleavage by thrombin, or by factor Xa, for activation; to subsequently inactivate the active form, proteolytic cleavage occurs at residue 336 by activated protein C or factor Xa. Activation is associated with cleavages between the A1 and A2 domains (position 372), between the A2 and B domains (position 740), and between the B and A3 domains (position 1689). The thrombin-activated form is referred to as factor VIIIa.sub.IIA and was believed to be a heterodimer with a subunit composition of A1/A3-C1-C2. Recently, Lollar et al., J. Biol. Chem. 266:12481 (1991); Fay, et al., J. Biol. Chem 266:8957 (1991); and Pittman, et al. Blood 79:389 (1992) presented evidence suggesting that human thrombin-activated VIII is heterotrimeric, i.e., A1/A2/A3-C1-C2.
Both human and porcine factor VIIIa.sub.IIA are unstable at physiological concentration and pH. However, porcine factor VIII has been found to be indefinitely stable at concentrations greater than 0.2 .mu.M, at pH 6.0; in contrast, human factor VIIIa.sub.IIA loses activity under these in vitro conditions. However, subjecting the thrombin activated human factor VIII to Mono S chromatography at pH 5.0 increases the activity 10 fold and the active fraction contains greater concentrations of the A2 domain. For both human and porcine factor VIIIa.sub.IIA, it has been suggested that dissociation of the A2 subunit is responsible for the loss of activity under the in vitro conditions selected (Lollar et al., supra.) However, no in vivo studies have been performed to ascertain whether the association/dissociation of the A2 subunit has any affect on the in vivo blood clotting activity of factor VIII.
DNA sequences for human factor VIII are known, as are expression methods. Toole, et al., Nature 312:312 (1984); Wood, et al., Nature 312:330; Vehar, et al., Nature 312:337 (1984) and WO 87/04187; WO 88/08035; and WO 88/03558. In addition, human factor VIII analogs have been developed to better understand the specific structural requirements for factor VIII activatibility, inactivatibility, and in vivo efficacy. Included among the features to be optimized are simplified preparation, ease of administration, stability, improved clearance/distribution characteristics, reduced immunogenicity, and prolonged half-life. Available analogs have modified the factor VIII structure such that part or all of the B domain has been deleted [U.S. Pat. No. 4,868,112] and/or specific amino acid positions are modified to reduce factor VIII susceptibility to cleavage at one or more sites [PCT/US87/01299 (WO87/07144)]. In addition, attempts have been made to replace the factor VIII B domain sequence with the B domain sequence of factor V, another cofactor involved in the coagulation cascade [U.S. Pat. No. 5,004,803].
In contrast to human factor VIII, elucidation of the complete nucleic acid sequence for porcine factor VIII has been hampered for many years. Partial amino acid information for porcine factor VIII was described in Church, et al., Proc. Natl. Acad. Sci. 81:6934 (1984) for an internal 98-residue segment; a 26-residue segment from the NH.sub.2 terminus and a 35-residue segment from the NH.sub.2 terminus of a 35-kD thrombin activation peptide. The actual sequence information disclosed was only of those porcine factor VIII sequences corresponding to the N-terminal sequence of the porcine light chain having homology to two structurally related proteins, i.e., coagulation factor V and ceruloplasmin. The 26-residue segment and 98-residue internal sequence were not correctly located. Data presented, infra, and those described by Toole, et al., Nature 312:312 (1984), demonstrate that the sequence assignment by Church, et al., Proc. Natl. Acad. Sci. 81:6934 (1984) was incorrect. Subsequently, Toole, et al., Nature 312:342 (1984) provided limited N-terminal amino acid sequence analysis of cleavage fragments of porcine factor VIII. This information proved unsuitable for designing probes useful in cloning porcine factor VIII cDNA in that the resultant probes were too highly degenerate and insufficiently specific to detect porcine factor VIII cDNA. Several years later, Toole, et al., Proc. Natl. Acad. Sci. USA 83:5939 (1986), reported a high degree of divergence of amino acid sequence between porcine and human B domains of the factor VIII; accordingly, the human sequence proved unsuitable for designing probes to clone porcine factor VIII cDNA.
To date no one has been successful in determining the full length sequence for porcine factor VIII. Without such information it has been impossible to determine which structural features, if any, may play a role in porcine factor VIII's increased stability. Moreover, without such information it has been impossible to construct hybrid, or chimeric, factor VIII molecules having increased stability and specific activity, as well as chimeric forms that are immunologically distinct and which may be useful to treat those patients that have developed antibodies to human factor VIII. Accordingly, there continues to exist a need for further structural information for porcine factor VIII to design factor VIII analogs having improved stability and specific activity.